The present invention relates to the BAFF receptor (“BAFF-R,” also known as BR3 and Ztnfr12), a member of the TNF family of receptor proteins. BAFF-R has been described in International Patent Publication WO 02/24909.
BAFF-R specifically binds the TNF family ligand, BAFF (also known as TALL-1, THANK, BLyS, neurokine α, TNSF13B, and zTNF4), which has been described in International Patent Publication WO 00/43032. BAFF enhances B cell survival in vitro (Batten et al. (2000) J. Exp. Med. 192(10): 1453-1466) and has emerged as a key regulator of peripheral B cell populations in vivo. it is believed that abnormally high levels of this ligand may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells (Batten et al., (2000) J. Exp. Med. 192(10): 1453-1466). Agonists and antagonists of BAFF activity have been described in WO 00/43032.
Recently, BAFF-specific agents, including BAFF antibodies, have been developed for treatment of autoimmune and other disorders (see, e.g., U.S. patent application Ser. Nos. 09/911,777; 10/380,703; 10/045,574; and 60/512,880); Kalled et al., (2003) Expert Opin. Ther. Targets, 7(1):115-23).
Prior to the discovery of BAFF-R, many members of the TNF receptor family had been uncovered by expressed sequence tag (EST) analysis and genomic sequencing. However, some family members, like BAFF-R, required expression cloning for their identification.
A member of the TNF receptor family, BAFF-R, is fairly divergent from many family members. In particular, BAFF-R contains only one cysteine-rich domain with 4 cysteines, while most TNF receptor family members typically contain 2-4 domains, each with 6 cysteines. This absence of a canonical receptor cysteine-rich domain prevented identification of BAFF-R by sequence-based searches. It was also not clear exactly how BAFF-R achieves high-affinity binding to BAFF, and exactly what sequences are involved.
Not only is BAFF-R distinct from the canonical TNF receptor family members, human BAFF-R (hBAFF-R) is only 60% homologous with murine BAFF-R (mBAFF-R). This difference is reflected in the differential aggregation of hBAFF-R (90% aggregated) and mBAFF-R (10% aggregated) when the extracellular domain is expressed in eukaryotic cells. However, two point mutations in hBAFF-R (V21N and L28P in SEQ ID NO:1) reduce its aggregation to less than 10%. This mutated form of hBAFF-R is referred to as vBAFF-R.
TNF family members are known to possess both N-linked and O-linked glycosylation sites. N-linked glycosylation occurs on asparagine residues within distinct consensus sequences. O-linked glycosylation occurs on serine and threonine residues, but a lack of sequence motifs and inconsistent addition of sugars within a population of proteins prevents the prediction of the presence of O-linked glycans on a protein. Additionally, O-linked glycosylation sites are often clustered in short serine/threonine-rich sequences, making it difficult to determine the exact number and location of the glycans. Even when a pattern of glycosylation can be determined for a specific protein, O-linked glycosylation is tissue dependent, so the pattern varies with the cell type in which the protein is being expressed.
The ability to analyze any given feature of a batch of protein product may be important for producing polypeptides for pharmaceutical use. The unpredictability and inconsistency of O-linked glycosylation leads to difficulties in manufacturing. For example, a large number of O-linked glycans makes it unfeasible to characterize batches of protein pharmaceuticals by mass spectrometry.
Possible solutions to this problem include the production of proteins in prokaryotic cells, which do not contain glycosylation machinery, and the production of proteins by chemical synthesis. However, glycosylated proteins may have advantages over non-glycosylated proteins. For example, O-linked glycosylation aids in folding and maintaining tertiary structure, causes increased stability and protease resistance, and modulates interactions with other proteins. O-linked glycosylation may also influence a protein's biological activity. It is known that the activity of many cell signaling molecules, including TNFα, is modulated by the glycosylation of cell surface receptors (Van den Steen et al. (1998) Crit. Rev. Biochem. Mol. Biol. 33(3): 151-208). Additionally, the production of proteins by chemical synthesis for pharmaceutical use can be prohibitively expensive.
Accordingly, a need exists to provide a BAFF-R protein for therapeutic use with a glycosylation pattern that can be characterized unambiguously, and that avoids aggregation in eukaryotic cells while maintaining specificity and affinity for BAFF.